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Western blot analysis of beta Actin on different lysates with Rabbit anti-beta Actin antibody (HA722023) at 1/20,000 dilution.
Lane 1: HeLa cell lysate (10 µg/Lane)
Lane 2: HepG2 cell lysate (10 µg/Lane)
Lane 3: MCF7 cell lysate (10 µg/Lane)
Lane 4: A431 cell lysate (10 µg/Lane)
Lane 5: Jurkat cell lysate (10 µg/Lane)
Lane 6: HEK-293 cell lysate (10 µg/Lane)
Lane 7: RAW264.7 cell lysate (10 µg/Lane)
Lane 8: C2C12 cell lysate (10 µg/Lane)
Lane 9: PC-12 cell lysate (10 µg/Lane)
Lane 10: Mouse testis tissue lysate (10 µg/Lane)
Lane 11: Mouse spleen tissue lysate (10 µg/Lane)
Lane 12: Rat testis tissue lysate (10 µg/Lane)
Lane 13: Rat kidney tissue lysate (10 µg/Lane)
Predicted band size: 42 kDa
Observed band size: 42 kDa
Exposure time: 3 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722023) at 1/20,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
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Western blot analysis of beta Actin on HepG2 cell lysates with Rabbit anti-beta Actin antibody (HA722023) at different dilutions.
Lysates/proteins at 20 µg/Lane.
Predicted band size: 42 kDa
Observed band size: 42 kDa
Exposure time: 30 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722023) at different dilutions was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
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Immunocytochemistry analysis of HeLa cells labeling beta Actin with Rabbit anti-beta Actin antibody (HA722023) at 1/250 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-beta Actin antibody (HA722023) at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Flow cytometric analysis of HeLa cells labeling beta Actin.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722023, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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Immunocytochemistry analysis of NIH/3T3 cells labeling beta Actin with Rabbit anti-beta Actin antibody (HA722023) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-beta Actin antibody (HA722023) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Flow cytometric analysis of NIH/3T3 cells labeling beta Actin.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722023, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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Immunocytochemistry analysis of L6 cells labeling beta Actin with Rabbit anti-beta Actin antibody (HA722023) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-beta Actin antibody (HA722023) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Flow cytometric analysis of C6 cells labeling beta Actin.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722023, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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beta Actin was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with HA722023 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722023 at 1/5,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: NIH/3T3 cell lysate (input)
Lane 2: HA722023 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of HA722023 in NIH/3T3 cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 2 seconds; ECL: K1801
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